Mechanistic and Synergistic Evaluation of Amodiaquine-Isometamidium Combination Against Trypanosoma evansi: Integrated In Silico-In-Vitro Insights.

backgroundTrypanosoma evansi (T. evansi) is the causative agent of surra, and it remains a serious threat to livestock health and productivity. This study aimed was to evaluate the trypanocidal potential of amodiaquine (AQ), alone and in combination with isometamidium chloride (IC), against T. evansi, using both in-silico and in-vitro testing.

methodsComputational testing involved induced-fit docking of AQ which trypanothione reductase (TR), followed by molecular dynamics (MD) simulations, and downstreampost-MD simulation analyses, intended to guide mechanistic hypotheses. In-vitro assays included drug sensitivity profiling across ten T. evansi isolates, checkerboard synergy testing, time–dependent kinetics, and qRT-PCR–based transcriptional analysis. Statistical tests included non-linear regression for IC90 estimation, one-way ANOVA and two-way ANOVA.

resultsMolecular docking revealed favorable AQ–TR interactions (Glide gscore –9.59 kcal/mol), further stabilized in MD simulations (RMSD 1.04 Å, RMSF 0.64 Å). MM-GBSA analysis yielded a favorable binding free energy (ΔG bind –27.52 kcal/mol). In-vitro, AQ had the IC90 values ranging from 4.27 to 69.29 µg/mL, whereas that of IC ranged from 4.49 to 31.01 ng/mL. Combination treatment significantly reduction in effective concentrations (AQ 8 µg/mL + IC 8 ng/mL vs. AQ 69.29 µg/mL, IC 31.01 ng/mL), with a synergistic FICI of 0.37 and complete clearance in high-dose time–kill assays. Upregulation of TR, TS, and TH under drug pressure was confirmed through qRT-PCR, which was the most pronounced with combination regimens (p < 0.05).

conclusionCollectively, these findings revealed AQ–IC co-administration is a mechanistically rational and pharmacodynamically synergistic strategy against drug-resistant T. evansi.